Metabolome analysis of genus Forsythia related constituents in Forsythia suspensa leaves and fruits using UPLC-ESI-QQQ-MS/MS technique

Forsythia suspensa is a traditional Chinese herb. Its numerous metabolites have important roles, as they possessed a wide range of biological activities. This study explored the accumulations of F. suspensa metabolites by performing widely targeted metabolomic analysis. The metabolites were studied at four stages of fruit development. Metabolites in the fruits and leaves of F. suspensa during fruit development included phenolic acids, flavonoids, lipids, lignans and coumarins, amino acids and their derivatives, terpenes, organic acids, nucleotides and their derivatives, alkaloids, quinones, steroids, and tannins. Fourteen Forsythia related metabolites were detected. Their contents varied among the developmental stages. Statistically significant correlations were found between the levels of forsythoside B and 11-methyl-forsythide, and forsythialan B and phillygenin, in both leaves and fruits. According to the correlation analysis between metabolites, Forsythia related metabolites were divided into two classes and five subclasses. In total, 33 compounds presented significant correlations in both fruits and leaves, which indicated the potential relationship in the synthesis of Forsythia related metabolites. Forsythialan B and phillygenin were both negatively correlated with L-valine, while Z-6,7-epoxyligustilid was positively correlated with both compounds. The quality control compounds forsythiaside A and phillyrin were positively and negatively correlated with uracil, respectively. These metabolomics results may facilitate the biosynthesis of Forsythia related metabolites.


Introduction
Forsythia suspensa (Tunb.) Vahl is widely used in medical applications [1][2][3][4]. Previous studies indicated that the compounds present in F. suspensa include phenylethanoid glycosides, analyzed the dynamics of Forsythia related medicinal metabolites in fruits and leaves to determine the optimum harvesting time and plant organ.

Plant material
The Forsythia suspensa plants were located in Jingxing, Shijiazhuang, Hebei, China. The leaves and fruits were sampled at four fruit developing stages in 2019. About twenty uniform asexual propagation seedlings of Forsythia suspensa were used for this study. We sampled fifty fruits which have relatively uniform growth and physiological state each sampling time as one biological replicate, and the fourth leaves were sampled with the same method. Each sampling was repeated three times to reduce the error and ensure measurement data validation and accuracy. The sample were collected around ten a.m. each time. After sampling, the samples were transported to the laboratory and cleaned in distilled water, and then put into liquid nitrogen and stored at −80˚C. We sampled the fruits and leaves every 50 days since May 30th. The sampling dates in 2019 were as follows, May 30th, July 20th, September 10th and October 30th. The four stages were as follows: early-stage (T1), mid-stage (T2), mid-late-stage (T3) and late-stage (T4).

UPLC and electrospray ionization triple quadrupole linear ion trap (ESI-Q TRAP-MS/MS) conditions
The sample extracts were analyzed using an UPLC-ESI-MS/MS system (UPLC, Shim-pack UFLC SHIMADZU CBM30A system, www.shimadzu.com.cn/; MS, Applied Biosystems 4500 Q TRAP, www.appliedbiosystems.com.cn/). The analytical conditions were as follows, UPLC: column, Agilent SB-C18 (1.8 μm, 2.1 mm 100 mm); The mobile phase was consisted of solvent A, pure water with 0.1% formic acid (HPLC grade, Merck, www.merckgroup.com/), and solvent B, acetonitrile (HPLC grade, Merck, www.merckgroup.com/). Sample measurements were performed with a gradient program that employed the starting conditions of 95% A, 5% B. Within 9min, a linear gradient to 5% A, 95% B was programmed, and a composition of 5% A, 95% B was kept for 1min. Subsequently, a composition of 95% A,5.0% B was adjusted within 1.10 min and kept for 2.9 min. The column oven was set to 40˚C; The injection volume was 4μl. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS. LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (Q TRAP), API 4500 Q TRAP UPLC/MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive and negative ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature 550˚C; ion spray voltage (IS) 5500 V (positive ion mode)/-4500 V (negative ion mode); ion source gas I (GSI), gas II(GSII), curtain gas (CUR) were set at 50, 60, and 30.0 psi, respectively; the collision activated dissociation (CAD) was set at high level. Instrument tuning and mass calibration were performed with 10 and 100 μmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scans were acquired as MRM experiments with collision gas (nitrogen) set to 5 psi. DP and CE for individual MRM transitions was done with further DP and CE optimization. A distincitve set of MRM transitions were monitored for each period according to the metabolites eluted within this period.

Correlation analysis of metabolites in leaves and fruits
The correlation analysis was performed with Forsythia related metabolites and other metabolites detected in this experiment from four fruit developing stages. The correlation values between Forsythia related metabolites and other metabolites were based on data of their ion intensities. All data were analyzed by bivariate analysis followed by Pearson's correlation and Two-tailed test of significance among different groups.
Experimental data of Forsythia related metabolites contents were analyzed by one-way analysis of variance followed by Tukey's multiple range test to detect differences among the groups. A p-value < 0.05 was considered significant. All statistical analyses were performed using IBM SPSS Statistics 22 software (IBM Corp., Armonk, NY, United States).

Analysis of levels of Forsythia related metabolites in leaves and fruits
In this study, ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was used to determine the fold-change values of Forsythia related metabolites in leaves and fruits. Quantifications utilized a QqQ mass analyzer. The total ion current (TIC) maps of the mixed sample (quality control sample, QC) were used to verify the reliability and repeatability of the experiment (S1 and S2 Figs). The peak shapes of the TIC maps were consistent, indicating that the results of the experimental results were reliable and repeatable. The extracted ion chromatograms (XIC) exhibited the chromatographic peaks corresponding to the mass metabolites (S3 and S4 Figs). The representative TICs an XICs of fruits and leaves at each harvesting time are shown in S5-S36 Figs. The mass spectrum information of Forsythia related constituents is shown in S1 Table. Forsythiaside C, forsythoside B, rengyoside B, rengyoside, forsythiaside B, forsythiaside J, forsythiaside A, isoforsythoside A, forsythialan B, forsythialan A, phillygenin, phillyrin, 11-methyl-forsythide and forsythide were all detected in fruits and leaves. The Forsythia related metabolites were classified as phenolic acids, lignans, and terpenoids, respectively. The fold-change values in the relative levels of metabolites in the fruits and leaves at different fruit developing stages varied markedly (Fig 1). The relative contents of forsythide, phillyrin, forsythiaside A, and isoforsythoside A were higher in leaves than in fruits, with the fold-change values < 1 at the four stages, which indicated that these constituents may be rich in leaves. In contrast, 11-methyl-forsythide, forsythenside B, forsythiaside J, forsythiaside C, forsythoside B, forsythialan A and forsythialan B might be the compounds rich in fruit. Forsythiaside J, forsythiashide A and forsythiaside C represented similar variation among the four fruit developing stages. Forsythiaside A in fruit exhibited lower levels than that in leaves, which differed from forsythiaside J and forsythiaside C. Phillygenin, forsythialan A and forsythialan B showed similar variations in fold-change values. However, phillygenin in fruits did not show much higher levels than that in leaves duing the fruit development process.

PLOS ONE
Analysis of genus Forsythia related constituents in leaves and fruits of Forsythia suspensa

Correlation analysis of Forsythia related metabolites
Fourteen metabolites were detected in the fruits and leaves of Forsythia. Correlation analyses revealed that forsythiaside C was positively correlated with forsythiaside J, and forsythoside B was positively correlated with 11-methyl-forsythide (Tables 1 and 2). Phillygenin was negatively correlated with rengyoside B, but was positively correlated with forsythialan A. In leaves, forsythiaside A, isoforsythoside A, and rengyoside A were not significantly correlated with any other Forsythia related metabolites.
Correlations were evident among metabolites in fruits. Forsythiaside C had significant positive correlations with forsythialan B, forsythialan, and phillygenin. Forsythoside B was positively correlated with 11-methyl-forsythide and forsythide. Forsythenside B was negatively correlated with both forsythiaside and isoforsythoside A, and positively correlated with phillyrin. Forsythiaside A was positively correlated with isoforsythoside A and negatively correlated with forsythenside B and phillyrin. Forsythialan A was positively correlated with forsythiaside C and forsythialan B; forsythoside B, 11-methyl-forsythide, and forsythialan B were all positively correlated with phillygenin in both leaves and fruits. However, forsythiaside J and rengyoside B were not significantly correlated with the other Forsythia related metabolites.
Despite the inconsistent correlations between Forsythia related metabolites in fruit and leaves. Forsythialan B, phillygenin and 11-methyl-forsythide and forsythoside B showed significant and corresponding positive correlations.
Based on the correlations shown in Figs 2 and 3, the Forsythia related metabolites were divided into two groups. Forsythiaside C, forsythenside B, forsythiaside J, forsythialan B, phillygenin, and phillyrin were all negatively or positively correlated with the other metabolites. However, the correlations between forsythoside B, rengyoside B, rengyoside A, forsythiaside A, isoforsythoside A, 11-methyl-forsythide, and forsythide and the other metabolites were inconsistent.
The Forsythia related metabolites and their substance types did not present any correlations. Phenolic acids were distributed in both classes, whereas only monoterpenoids and lignans were present in two classes. The correlations between metabolites of the same substance type and Forsythia related metabolites were inconsistent.
When ranked by forsythiaside, heatmaps of correlation values of Forsythia related metabolites showed a similarity of correlations between metabolites in fruits and leaves (Fig 3). Correlation values between other metabolites and Forsythia related metabolites in different plant organs exhibited similar results. These findings indicated the reproducibility of the potential metabolic pathways and the differentiation of metabolites. The results suggested that the metabolic pathways of Forsythia related metabolites could be divided into different groups.

Molecular structures and cluster analysis of Forsythia related metabolites
To further explore the relationships between the Forsythia related metabolites and other metabolites, a cluster analysis was performed (Fig 3). The Forsythia related metabolites could be divided into two classes, which were each further divided into two subclasses. Forsythia related metabolites in the first subclass of class 1 were forsythialan B, phillygenin, forsythiaside C, phillyrin, forsythiaside J, and forsythenside B. Only forsythialan A was clustered in the second class. The Forsythia related metabolites in the first subclass of the class 2 included rengyoside B, forsythiaside A, forsythoside B, 11-methyl-forsythide, rengyoside A, and forsythide. Only isoforsythoside A was clustered in the second subclass. The Forsythia related metabolites in classes 1 and 2 were lignans, phenolic acids, monoterpenoids, and phenolic acids. (Table 3, Fig 4).
In the second subclass of Forsythia related metabolites, isoforsythoside A, forsythoside B, rengyoside A, forsythiaside A, rengyoside B, forsythide and 11-methyl-forsythide all displayed the same structure as (2R,3R,4S,5S,6S)-tetrahydro-2H-pyran-2,3,4,5,6-pentaol (Figs 4 and 5). Although 11-methyl-forsythide and forsythoside B were in the same cluster, but 11-methylforsythide was more likely to be a derivative of forsythide. In addition, methylation was observed in the molecular structures of 11-methyl-forsythide and forsythoside B. Methylations also occurred in the cluster of rengyoside B and forsythiaside A as well. Although forsythiaside A and isoforsythoside A have the same groups, cluster analysis revealed a distant relationship. However, isoforsythoside A also undergoes methylation.

Discussion
In this study, Forsythia related metabolites in leaves and fruits during different fruit development stages were analyzed. The goal was to clarify the optimal harvesting time necessary to achieve a high content of medicinal compounds, and to establish the plant organs with the highest metabolite contents. Forsythia suspensa has multiple medical applications because of its complex mixture of metabolites in leaves and fruits [25][26][27]. Significant differences in plant metabolic processes among plant organs have been described. Studies on F. suspensa have challenged the belief that the fruit is the main source of medicinal compounds, with leaves reportedly being a better source [26]. In the present study, the levels of phillyrin, forsythiaside A, isoforsythoside A, and forsythide in leaves were higher than those in fruits, consistent with the results of some previous studies, whereas the levels of forsythenside B, forsythoside B, forsythialan A, forsythialan B, and forsythiaside J were higher in fruits than in leaves. These compounds derived from leaves may be less suitable for medical use. The levels of the Forsythia related metabolites differed among development stages and plant organs, which is an important consideration for harvesting.
Isoforsythoside A and forsythiaside A have been reported to have inhibitory effects on Staphylococcus aureus and other pathogenic bacteria [31]. One study indicated that these molecules also have potent activities against fungi and influenza A virus [32]. Forsyhia suspensa metabolites also have antiseptic properties. Forsythin has been reported to exhibits anti-oxidation activity, lowers blood lipid levels and inhibits the oxidation of low-density lipoprotein [33]. Another study demonstrated that phillyrin can enhance the scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) free radicals [34]. The present study confirmed differences in the content of Forsythia related metabolites between leaves and fruits. Different therapeutic effects can be expected between compounds derived from the leaves and fruits of F. suspensa. The plant organs from which compounds are extracted and the harvesting stage should be considered to ensure that the desired medicinal effects are achieved. We suggest that the leaves of Forsythia suspensa harvested at an intermediate stage, would likely provide the greatest medicinal effects because of their high levels of forsythiaside A, isoforsythoside A, phillygenin, and phillyrin.
Significant progress has been made in determining the components of medicinal herbs with medical applications, and the biosynthetic pathways of podophyllotoxin and colchicine have been identified [35,36]. Studies on colchicine and podophyllotoxin have indicated that a combined metabolomic and transcriptomic analysis approach is suitable for studying biosynthetic pathways. Metabolomics is the major tool for the identification and screening of target substances, and determination of their levels in different plants. However, the biosynthetic pathways in F. suspensa are still not fully understood. Although previous studies have involved only enzyme assays, metabolic research offers much promise as a means of understanding the basis of biosynthetic pathways. A previous transcriptome study of the biosynthesis pathways of lignans performed in Forsythia koreana revealed that pinoresinol-lariciresinol reductase (PLR) and matairesinol O-methyltransferase (MOMT) from this plant catalyzed the biosynthesis of lariciresinol from pinoresinol and biosynthesis of larctigenin from matairesinol [37]. Phillygenin, forsythialan A and forsythialan B were all classified into one cluster by analyzing their correlations. PLR and MOMT regulate the transformations of pinoresinol to lariciresinol and of matairesinol to arctigenin, which are similar to the transformation of phillgenin to forsythialan A and from forsythialan A to forsythialan B. Thus, PLR and MOMT may be involved in the biosynthesis of distinctive metabolites of Forsythia. Because PLR and MOMT of F. koreana participate in the biosynthesis processes of forsythialan A and forsythialan B, we assumed that forsythialan A and forsythialan B are the downstream metabolites of phillygenin. We suggest that the medicinal functions of phillygenin, forsythialan A and forsythialan B should be further studied to verify the functions of PLR and MOMT in Forsythia suspensa. Ying et al. [38] reported the different catalytic efficiencies and specificities of pinoresinollariciresinol reductases and pinoresinol reductases (PrRs) in Arabidopsis thaliana and Isatis indigotica by analyzing and comparing the crystal structures of PLRs and pinoresinol reductases. The findings indicated that the different crystal structures affect the catalytic efficiencies and selectivities. PLR1 from I. indigotica and A. thaliana reduce pinoresinol and lariciresinol with different catalytic efficiencies, wherease pinoresinol reductase 2 from A. thaliana only pinoresinol to produce lariciresinol. Thus, the crystal structures of PLRs in different species might be different, which results in different metabolism of lignans and the production of differing amounts of a variety of active substances [38,39]. We assume that the PLRs in Forsythia may from A. thaliana reduces have important roles in the biosynthesis of Forsythia specific metabolites. The metabolic functions of PLRs in Forsythia need to be further studied. Our study revealed positive correlations between forsythoside B and 11-methyl-forsythide and forsythialan B and phillygenin, in both leaves and fruits. Thus, there might be commonalities among these metabolites in terms of their biosynthetic pathways and precursors. Cluster analysis of the metabolites indicated that the two major classes identified may be subject to similar biosynthetic processes. Phenolic acids were present in both classes, whereas lignans and terpenoids were present in two different subclasses respectively. Correlation analysis revealed both positive and negative correlations among metabolites, but only a few of these significant or highly significant. Consistent correlations among Forsythia related metabolites within a single class indicated that their biosynthetic pathways may have similar processes in upstream metabolism. The chemical structures of the metabolites in each subclass further indicated the differentiation of the biosynthetic pathways. Although the chemical structures of many metabolites have already been identified, their biosynthetic pathways remain poorly understood. Phenolic acids presented in both classes, and their biosynthetic processes may play important roles in their upstream metabolism. Terpenoids and lignans were clustered into different subclasses separately. The biosynthetic pathways of the lignans and terpenoids may have significant effects on metabolites differentiation. The biosynthetic pathways of the Forsythia related metabolites were significantly different, and there might be different upstream biosynthetic processes in the two subclasses. Differences in synthesis and differentiation of downstream products may underlie the different metabolite compositions of the two classes. In the present study, forsythialan B and phillygenin, 11-methyl-forsythide and forsythoside B showed negative or positive correlations, respectively. The cluster analysis also showed that forsythialan B and phillygenin 11-methyl-forsythide and forsythoside B may have similar processes in their biosynthesis pathways, which also confirmed the validity of the correlation and cluster analysis. Forsythialan B, phillygenin, forsythiaside C, forsythiaside J, forsythiaside B, phillyrin and forsthialan A were clustered in one subclass. However, forsythialan B, phillygenin, phillyrin and forsythialan A were identified as lignans. Although the other metabolites were phenolic acids, and phenolic acids present in both subclasses. Thus, we suggest that the studies on metabolites of these lignans should focus on the transformations from phenolic acid to lignans and the differentiations of their chemical groups, such as methoxy and hydroxyl. Forsythide and 11-methylforsythide were monoterpenoids, and clustered into one subclass. Their chemical structures and cluster results showed similarities in biosynthesis processes. Enzymes that catalyze the insertion of the ether bond may facilite the biosynthesis of 11-methylforsythide. In addition, phillygenin was identified to play an important role in the biosynthesis pathways of the Forsythia related metabolites, and hydroxylationrelated genes and enzymes may be involved in the processes of forsythialan A and forsythialan B. Tetrahydro-1H,3H-furo [3,4-c]furan was a common group of the Forsythia related metabolites in the first subclass. Forsythia related metabolites of the second subclass did not have similar furan groups. Thus, we assume that the biosynthesis and insertion of tetrahydro-1H,3H-furo [3,4-c]furan may occur upstream. (2R,3R,4s,5S,6S)tetrahydro-2H-pyran-2,3,4,5,6-pentaol, was found in most of the Forsythia related metabolites as a common group. Its biosynthesis may occur upstream, as there was no indication of its formation in the differentiation process of the Forsythia related metabolites. As only ether bond of the common group, (2R,3R,4s,5S,6S)-tetrahydro-2H-pyran-2,3,4,5,6-pentaol, of 11-methylforsythide was replaced with a methylene. We assume that compared with other Forsythia related metabolites, 11-methylforsythide was a downstream metabolite. As phillyrin had both of two common groups, its biosynthesis may be a cross procedure among the biosyntheses of the two subclasses of Forsythia related metabolites. Cluster analysis and molecular structures indicated that there are the potential biosynthesis orders of the Forsythia related metabolites.
In this study, we analyzed the Forsythia related constituents in leaves and fruits at different harvesting stages. The findings support the suggestion that harvesting should be timed with the metabolites contents. The stability and differences in metabolism of forsythiaside A and phillyrin levels during fruit development could facilitate the identification of the two classes of Forsythia related metabolites reported in this study. This confirms the rationality of the quality evaluation method for F. suspensa in the Chinese Pharmacopeia. This study provides a theoretical basis for further study of the Forsythia related metabolites in F. suspensa, including in terms of biosynthesis and metabolic pathways.

Conclusions
Different metabolites levels and proportions of metabolites may influence the medicinal properties of F.suspensa. We found that the metabolites levels in both fruits and leaves varied during different developmental periods. Thus, the harvest timing and plant organs should be carfully considered. Correlation and cluster analyses indicated two major types of potential biosynthesis pathways of the Forsythia related metabolites in F.suspensa. (2R,3R,4s,5S,6S)-Tetrahydro-2H-pyran-2,3,4,5,6-pentaol and phillygenin are two typical molecular structures of Forsythia related metabolites. The related enzymes and genes involved in the insertion of (2R,3R,4s,5S,6S)-tetrahydro-2H-pyran-2,3,4,5,6-pentaol may play important roles in the formation of both types of Forsythia related metabolites. Related hydroxylation enzymes and hydrogenases may be involved in the differentiation of metabolites into the first type.